GSM7839297: sc-RNA seq S6; Rattus norvegicus; RNA-Seq
A modified Smart-seq2 protocol developed by the Tang laboratory was used for single-cell RNA-seq. Briefly, acutely dissociated DRG neurons were plated on dishes as described previously. After 15 min, the DMEM was replaced with PBS, and a mouth pipette was then used to place each individual fluorescently labeled single DRG neuron into a volume of lysis buffer under a fluorescence microscope. Harvesting of the single DRG neurons from one rat was completed in 4 h, and the L4-5 DRG of at least 5 rats were harvested at each time point. The reverse transcription reaction was performed with a TSO primer added at the 5' end and with 25 nt oligo (dT) primer anchored with an 8 nt cell-specific barcode and 8 nt unique molecular identifiers (UMIs) added at the 3' end. After reverse transcription, cDNAs were amplified by 15 cycles of PCR, during which the IS primer and the 3'P2 primer were added to the 5' and 3' ends of the cDNAs, respectively. The amplified cDNAs from single cells were pooled, purified using a DNA Clean & concentration-5 kit (Zymo Research, D4014) and AMPure XP beads (Beckman Coulter, A63882), and quantified using a Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). To add biotin tags to the 3' ends of the amplified cDNAs, biotinylated preindexed primers and IS primer were used to further amplify the PCR product through an additional 4 cycles of PCR. The biotinylated cDNAs were then sheared to approximately 300 bp using Covaris S2, and the 3' termini of the cDNAs were captured using C1 beads (Dynabeads® Myone™ Streptavidin C1, Invitrogen, 65001). The RNA-seq library was constructed using a Kapa Hyper Prep Kit (KAPA Biosystems, KK8505), and short universal primers and QP2 primers were used in the last amplification. Finally, the genome and transcriptome libraries were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (sequenced by Novogene).